The generation of induced pluripotent stem cells (iPSC) from adult somatic cells affords a novel approach the study and potential treatment of genetic diseases on a patient-specific level. Recently, our laboratory and others have pioneered methods for generating differentiated hepatic cells from stem cells (ES or iPS). Familial hypercholesterolemia (FH) is a common (1:500) inherited hyperlipidemia caused by mutations in the LDL receptor (LDLR). Patients exhibit markedly elevated LDL in the presence of normal TG and HDL, and experience cardiovascular sequelae at an early age. As the central regulator of lipid homeostasis and the primary source of clearance of LDL from the serum, the liver is central to the pathogenesis of FH. Statins and other effective pharmacotherapies exert their lipid-lowering effects at the hepatocyte level, and liver transplant results in complete resolution of disease in homozygous FH patients. In light of the recent advances in iPSC generation, hepatic cell derivation from human ES/iPS cells, and the significant impact that would be achieved through greater understanding of hyperlipidemias and heart disease, we hypothesized that stem cell-derived hepatocytes created using iPSCs from patients with FH would recapitulate the hepatocyte-specific pathology that defines the disease, providing a novel in-vitro model for further study of FH pathology, its pharmacologic modulation, and development of additional strategies for its management. We sought to validate such a model, providing a proof-of-principle regarding iPSC-derived hepatocytes and disease modeling, specifically with regard to dyslipidemias. Methods: We generate iPSC lines from human fibroblasts by lentiviral transduction (vectors encoding Oct4, Nanog, Sox2, Lin28) and validate the resulting iPSC by immunostaining/fluorescence microscopy, flow cytometry, DNA fingerprint STR analysis, karyotyping, and teratoma assay. Directed differentiation of iPSCs to hepatic cells involves a 20-day, 4-step protocol optimized and recently published by our laboratory. The characterization of the lipid-metabolic phenotype of iPSC-derived hepatocytes from normal and FH individuals will involve a multi-dimensional and multi-tool approach. We use both a BAC-based targeting approach for correcting FH iPSC's, and a somatic gene therapy approach using AAV-vector based gene delivery to iPS-derived hepatocytes. We will evaluate the functional rescue of the FH phenotype following correction. Significance and Conclusion: The demonstration that FH iPSC-derived hepatocytes reproduce key features of the pathology of the disease would represent an important proof-of principle regarding disease modeling using patient-specific iPSC-derived hepatocytes. Based on our discovery that FH iPS-derived hepatocytes secrete VLDL at nearly 10-fold the rate of 3 disparate WT controls, we believe that this novel system may resolve or at least contribute to the understanding of the 'ApoB paradigm'in the pathogenesis of FH. Last, our 2 targeting approaches address separate, critical applications of iPS-derived tissues: 1) whether iPS can be corrected without disrupting endogenous genes followed by differentiation and rescue of phenotype, paving the way for possible creation of patient-specific transplants, and 2) whether iPS-derived hepatocytes can serve as a model for testing and refining the efficacy of the promising AAV class of vectors for somatic gene targeting in vivo, which would significantly accelerate their usefulness as clinical therapies. PUBLIC HEALTH RELEVANCE: In the proposed project we will generate multiple induced pluripotent stem cell lines from patients with familial hypercholesterolemia, a disorder caused my mutations in the LDL receptor gene, which we will differentiate into hepatocytes in order to study the pathology of the disease and test 2 approaches for genetically rescuing the phenotype in vitro.